Kinetic Studies on Na⁺/K⁺-ATP_ase and Inhibition of Na⁺/K⁺-ATP_ase by ATP

Abstract

Na⁺/K⁺-ATP_ase (EC 3.6.1.3) is an important membrane-bound enzyme. In this paper, kinetic studies on Na⁺/K⁺-ATP_ase were carried out under mimetic physiological conditions. By using microcalorimeter, a thermokinetic method was employed for the first time. Compared with other methods, it provided accurate measurements of not only thermodynamic data (δ_rH_m) but also the kinetic data (K_m and V_max). At 310.15 K and pH 7.4, the molar reaction enthalpy (δ_rH_m) was measured as -40.514 ± 0.9 kJ mol⁻¹. The Michaelis constant (K_m) was determined to be 0.479 ± 0.020 mM and consistent with literature data. The reliability of the thermokinetic method was further confirmed by colorimetric studies. Furthermore, a simple and reliable kinetic procedure was presented for ascertaining the true substrate for Na⁺/K⁺-ATP_ase and determining the effect of free ATP. Results showed that the MgATP complex was the real substrate with a K_m value of about 0.5 mM and free ATP was a competitive inhibitor with a K_i value of 0.253 mM.

Key Words:

• Na⁺/K⁺-ATP_ase
• Microcalorimeter
• Thermokinetic method
• Competitive inhibitor
• Kinetics